Project Summary Secreted extracellular vesicles (EVs) may play a role in biological processes and disease pathogenesis. Impact of these EVs on Human Immunodeficiency Virus type 1(HIV-1) infection has only recently started to be investigated. In fact, EVs such as exosomes have been shown to influence cells within the central nervous system (CNS) and modulate immune responses to pathogens. The HIV Negative factor (Nef) is released from nef-transfected or HIV-infected immune cells in exosomes, extracellular nano-sized vesicles generally used for para- or intercellular communication ? delivery of antigen, modification of gene expression, and modulation of immune responses. Interestingly, microglia infected with HIV or transfected with a nef-gfp expression plasmid release Nef in exosomes. However, the role this extracellular exosomal Nef (exNef) may have in HIV replication within the CNS and neuropathogenesis is unknown. It is known that HIV infects cells within the brain, persists within the CNS despite successful combination anti-retroviral therapy (cART), and causes neurocognitive impairments such as HIV-associated Neurological Disorder (HAND). Although cART significantly lowers peripheral viral load to undetectable levels(aviremia), HAND is still observed in among 40% of virally suppressed HIV+ individuals. Together these findings suggest that in the presence of cART a novel mechanism not associated with the HIV is at play to induce neurocognitive impairment. Substance abuse could also play a key role in HIV disease progression and the onset of neurocognitive impairment. Opiates such as heroin, and its active metabolite morphine have been shown to increase the rate of HIV disease progression to NeuroAIDS and increase both the risk and severity of HAND in people living with HIV/AIDS (PLWHAs). Given that almost one- third of PLWHAs report heroin abuse, it is important to understand how opiates and cART interplay in HIV disease to cause neurocognitive impairment in order to improve the quality of life for these HIV+ individuals. We hypothesize that opiate-induced modifications in Nef+ EV composition and release exacerbates exNef associated neuronal damage and leads to greater neurocognitive impairment in the context of cART. In this proposal we will investigate in the context of cART and opiates, the impact of extracellular vesicles, specifically exNef released from HIV-infected (or nef- transfected) microglia on neurons in order to understand the mechanism(s) that underlie HIV-induced neurocognitive impairment/HAND during aviremia. We will also perform a cross-sectional study comparing cerebral spinal fluid(CSF) exNef in PLWHAs on cART with a history/current opiate use and neurocognitive impairment/HAND. Findings from this proposal will allow us to demonstrate the role of EVs in the neuropathogenesis induced by the interplay of opiates and HIV in the CNS.